Table 1: Top 31 ligands from virtual screening of CB306 vs P. vivax STPP 2B.
Score
S(PLP)
S(hbond)
S(cho)
S(metal)
DE(clash)
DE(tors)
intcor
time
Ligand name
MW
Hdon
Hacc
LogP
84.35
-33.49
1.17
0
8
0
0.33
0.04
22.019
'5467784'
228.25
2
2
2.3
83.11
-50.09
2
0
4.94
0
1.35
0.06
54.924
'7746693'
357.76
0
5
5
79.71
-40.64
1.32
0
5.99
0.11
0.39
0.06
69.394
'5282931'
316.48
3
4
5.1
78.85
-23.92
5.67
0.95
5.99
0
0.46
0.03
19.496
'5175515'
367.25
0
3
4.8
78.85
-18.56
1.58
0
9.96
0
2.23
0.13
58.665
'5192853'
333.45
0
4
2.5
76.55
-36.98
1.68
0
5.97
0
0.66
0.01
37.735
'5101823'
376.81
0
4
3.3
76.16
-42.25
1.76
0
4.98
0
0.63
0.01
71.36
'7591231'
385.46
3
6
0.4
75.23
-30.61
4.84
0
6
3.28
1.38
0.13
69.687
'7726180'
384.81
1
6
2.5
74.74
-37.28
2.86
0
4.98
0.11
0.47
0.02
13.167
'7437091'
311.4
2
2
2.7
74.43
-32.5
2.2
0
6
0
0.42
0.04
82.893
'6407567'
363.45
1
4
4.6
74.3
-42.62
1
0
4.97
0
0.61
0.07
20.259
'7724431'
366.41
0
5
4.5
74.23
-19.12
2.94
0
7.93
0
0.64
0.01
39.471
6433056'
72.81
-33.01
1.7
0
5.94
0
1.5
2.08
15.489
'7676009'
72.71
-32.07
1.88
0
6
0
0.52
0.03
47.624
'7842136'
71.42
-34.02
0.79
0
6
0
0.49
0.03
49.983
'7397377'
70.89
-37.94
1.63
0
4.94
0
0.8
0.02
40.391
'7761278'
70.72
-31.67
3.32
0
4.98
0
0.71
0.62
55.11
'5256974'
70.2
-29.58
1.74
0
5.99
0
1.41
2.25
54.344
'6629394'
70.17
-34.44
0
0
6
0
0.15
0.03
20.739
'5490779'
70.13
-30.04
1.64
0
5.88
0
0.06
0
42.055
'7814264'
70.01
-44.42
4.61
0
2
0
0.13
0.01
33.793
'7679150'
69.48
-34.72
0
0
6
0
0.64
0.04
29.375
'6565654'
69.48
-28.04
2
0
5.96
0
0.18
0.02
32.623
'7907769'
68.97
-38.96
0.75
0
4.93
0
0.95
0.01
13.549
'7589277'
68.9
-30.61
1
0
5.97
0
0.28
0.01
39.715
'5135469'
68.84
-41.39
0
0
4.91
0
1.03
0.04
51.657
'7928256'
67.99
-27.32
1.62
0
6
0
0.1
0.01
22.487
'9040855'
67.96
-49.19
0.96
0
2.91
0
0.8
0.03
53.728
'9027311'
67.91
-45.37
2.01
0
3
0
0.76
0.06
59.046
'7957875'
67.86
-33.79
0.03
0
6
0
1.02
0.01
10.964
'7278181'
67.68
-42.92
2.46
0
2.99
0
0.59
0.55
67.731
'5525637'
67.66
-35.66
1
0
4.97
0
0.41
0.01
49.641
'7903592'
Table 2: Top 45 ligands from virtual screening of NIHClinicalCollection vs P. vivax STPP 2B.
Score
S(PLP)
S(hbond)
S(cho)
S(metal)
DE(clash)
DE(tors)
intcor
Ligand name
MW
Hdon
Hacc
LogP
131.95
-90.39
4.35
0
5.21
0.18
5.39
8.2
'SAM001246803'
880.98
4
12
93.75
-29.77
4.86
0
8.76
0
2.32
0.88
'SAM001246552'
380.43
3
7
91.28
-39.42
0.94
0
8.59
0
1.44
0.19
'SAM001246721'
317.42
2
3
3.2
88.51
-70.27
1
0
2.81
0
1.5
1.3
'SAM001247096'
415.57
4
5
3.9
88.29
-74.04
1.65
0
2
0
1.78
0.64
'SAM001246653'
506.47
1
5
87.64
-35.35
2.86
0
7.95
0
2.42
0.52
'SAM001246684'
348.39
3
6
-0.7
86.24
-73.57
2.06
0
2
7.59
5.01
12.07
'SAM001246784'
1329.46
18
18
85.52
-49.49
6.43
0
3
0.03
1.02
0.58
'SAM001246528'
610.52
10
16
-1.3
85.27
-49.78
0.36
0
5.94
0
0.9
0.34
'SAM001246575'
388.89
1
5
1.7
84.97
-24.38
0.51
0
9.95
0
0.31
0
'SAM001246904'
372.41
0
5
84.53
-41.57
0
0
7.97
0
3.43
1.99
'SAM001246546'
83.65
-54.77
6
0
2
0
0.75
0.36
'SAM001246840'
83.56
-59.04
3
0
3
0
1.55
0.65
'SAM001246736'
83.55
-42.25
2.41
0
6
0
2.67
3.4
'SAM001246554'
83.47
-44.46
2.38
0
6
0
2.22
0.34
'SAM001246985'
82.69
-31.5
0
0
8.63
0
1.55
2.53
'SAM001246635'
82.37
-63.75
2.99
0
2
0
1.4
0.46
'SAM001246618'
81.88
-46.11
0.27
0
6
0
0.63
0.11
'SAM001246695'
80.84
-68.24
1
0
2
0
1.25
0.11
'SAM001246863'
80.62
-30.59
1.78
0
8.98
6.44
1.56
0.32
'SAM001246806'
80.56
-54.58
0.85
0
3.99
0
1.82
3.15
'SAM001246602'
80.11
-42.34
0.38
0.84
6
0.09
1.91
1.99
'SAM001246625'
79.48
-41.19
3.01
0
4.94
0
1.46
2.56
'SAM001246818'
78.7
-49.7
4.87
0
2.94
0
2.06
0.66
'SAM001246560'
78.6
-45.26
6.61
0
3
0
2.51
0.2
'SAM001246852'
77.84
-40.82
1.06
0
5.94
0
0.9
0.02
'SAM001246593'
76.44
-60.65
1
0
2.84
0
2.37
0.35
'SAM001247090'
76.07
-60.72
1.58
0
2.21
0
1.84
0.91
'SAM001247084'
76.03
-77.09
1
0
0
0
4.48
4.9
'SAM001246793'
75.83
-32.33
2.98
0
6
0.34
2.07
3.06
'SAM001246530'
75.43
-55.1
3.59
0
2
0
2.12
1.81
'SAM001246534'
75.16
-41.07
0.9
0
5.99
0
3.83
3.1
'SAM001246816'
74.98
-57.81
1
0
3
1.41
2.76
3.1
'SAM001246710'
74.84
-34.16
1.98
0
5.99
0
0.62
0.01
'SAM001246628'
74.35
-79.58
1.8
0
0
4.26
4.75
3.14
'SAM001246783'
74.1
-61.83
5.51
0
0
0
3.12
1.97
'SAM001246657'
73.99
-52.67
3.23
0
2
0
0.79
1.22
'SAM001246591'
73.67
-48.33
3.01
0
2.99
0
0.95
0.28
'SAM001246638'
73.24
-59.11
0.65
0.83
2
0
1.25
0.21
'SAM001247045'
72.72
-34.97
6.98
0
3
0.02
0.98
0.84
'SAM001246767'
72.39
-50.12
4.07
0
2
0
1.23
0.41
'SAM001246533'
72.33
-31.13
2.17
0
5.97
0
0.66
0.17
'SAM001246804'
71.59
-60.6
0
0
2
0
0.59
0.18
'SAM001246970'
71.36
-48.04
0.27
0
3.99
0
0.81
0.17
'SAM001246988'
71.15
-50.16
1.96
0
3
0
1.88
0.58
'SAM001246671'
Table 3: Top 40 ligands from virtual screening of InHouseCompounds library vs. P. vivax STPP2B.
Score
S(PLP)
S(hbond)
S(cho)
S(metal)
DE(clash)
DE(tors)
intcor
Ligand name
MW
Hdon
Hacc
LogP
82.55
-23.82
0.25
0
9.98
0
0.94
0.01
'5107893'
565.03
1
6
3.8
75.7
-50.56
0.85
0
3.99
0
0.83
0.25
'7796224'
395.43
1
5
3.8
72.78
-55.06
0.89
0
2.84
0
1.02
0.05
'6703891'
758.04
3
7
10.1
72.44
-48.11
0.9
0
3.9
0
1.14
0.37
'7752888'
412.4
0
8
3.7
70.41
-42.9
2.46
0
3.7
0
1.22
0.27
'7706580'
393.5
1
6
3.8
69
-54.93
0.69
0.44
1.99
0
0.7
0.11
'7785278'
283.32
1
3
3.3
67.04
-40.04
3.43
0
3
0
1.63
1.97
'7803112'
369.14
1
5
1.7
66.9
-38.5
2.23
0
3.97
0
1.1
0.07
'7186658'
394.42
1
6
3.6
64.67
-45.05
3.07
0
2
0
0.83
0.08
'5153298'
210.23
1
4
1.9
64.4
-42.42
1
0.43
3
0
0.29
0.3
'5380289'
265.39
2
2
3.7
63.32
-56.42
0.68
0
1
0
0.73
0.31
'7833474'
62.54
-50.79
0.99
0
1.99
0
1.67
0.21
'7646744'
62.4
-47.49
1.59
0
1.97
0
0.86
0.01
'7811263'
61.22
-53.84
0.93
0
1
0
0.78
0.06
'7637680'
60.47
-35.35
3.39
0
2.99
0.08
1.65
0.42
'5587264'
60.31
-40.27
2.94
0
2
0
0.49
0.2
'7748642'
59.74
-43.1
1.94
0
1.99
0.09
0.55
0.1
'5470088'
59.42
-42.55
2.63
0
1.94
0
1.38
0.12
'7733855'
59
-44.15
1.46
0
1.94
0
0.66
0.05
'5915863'
58.04
-41.6
2
0
2
0
0.81
0.07
'5153298'
57.6
-46.88
1.57
0.76
1
0
1.2
0.15
'6060460'
57.42
-59.34
0
0
0
0
3.07
4.21
'5115900'
57.26
-23.84
3.44
0
3.97
0
0.45
0.2
'5584856'
55.63
-31.1
2.37
0
2.99
0
1.24
1.98
'5648649'
54.82
-38.2
1.69
0
2
0
1.23
2.02
'5852635'
54.47
-53.89
0.99
0
0
0
1.38
0.37
'7477596'
54.2
-36.08
2.73
0
2
0
1.13
0.16
'5753084'
53.94
-43.47
0.67
0
1.69
0
1.03
0.22
'5753084'
53.9
-40.29
3.44
0
0.94
0
1.28
0.11
'5153298'
53.79
-36.72
1.7
0
2
0
1.02
2.01
'5852635'
53.7
-48.05
1.64
0.88
0
0.12
0.97
0.07
'7911340'
53.2
-24.78
1.96
0
3.99
0
2.02
2.6
'7923489'
52.66
-51.44
0.84
0
0
0
0.72
0.09
'5915863'
51.05
-52.55
0
0
0
0
1.07
0.63
'5128798'
50.76
-29.09
1.58
0
2.88
0
1.69
3
'5546306'
49.66
-47.35
1.64
0
0
0
2.62
2.63
'7680193'
49.26
-50.77
0
0
0
0
0.78
0.06
'5145897'
46.79
-46.63
1.61
0
0
0
4.33
4
'5163673'
44.61
-45.67
0
0
0
0
0.61
0.15
'5143459'
43.34
-45.78
0
0
0
0
2.52
2.59
'7542998'
Table 4: Best ranking list for all libraries screened against P. vivax STPP 2B homology model.
Score
S(PLP)
S(hbond)
S(cho)
S(metal)
DE(clash)
DE(tors)
intcor
Ligand name
MW
Hdon
Hacc
LogP
Lipinski?
131.95
-90.39
4.35
0
5.21
0.18
5.39
8.2
'SAM001246803'
880.98
4
12
No
93.75
-29.77
4.86
0
8.76
0
2.32
0.88
'SAM001246552'
380.43
3
7
Yes
91.28
-39.42
0.94
0
8.59
0
1.44
0.19
'SAM001246721'
317.42
2
3
3.2
Yes
88.51
-70.27
1
0
2.81
0
1.5
1.3
'SAM001247096'
415.57
4
5
3.9
Yes
88.29
-74.04
1.65
0
2
0
1.78
0.64
'SAM001246653'
506.47
1
5
No
87.64
-35.35
2.86
0
7.95
0
2.42
0.52
'SAM001246684'
348.39
3
6
-0.7
Yes
86.24
-73.57
2.06
0
2
7.59
5.01
12.07
'SAM001246784'
1329.46
18
18
No
85.52
-49.49
6.43
0
3
0.03
1.02
0.58
'SAM001246528'
610.52
10
16
-1.3
No
85.27
-49.78
0.36
0
5.94
0
0.9
0.34
'SAM001246575'
388.89
1
5
1.7
Yes
84.97
-24.38
0.51
0
9.95
0
0.31
0
'SAM001246904'
372.41
0
5
Yes
84.53
-41.57
0
0
7.97
0
3.43
1.99
'SAM001246546'
84.35
-33.49
1.17
0
8
0
0.33
0.04
'5467784'
228.25
2
2
2.3
83.65
-54.77
6
0
2
0
0.75
0.36
'SAM001246840'
83.56
-59.04
3
0
3
0
1.55
0.65
'SAM001246736'
83.55
-42.25
2.41
0
6
0
2.67
3.4
'SAM001246554'
83.47
-44.46
2.38
0
6
0
2.22
0.34
'SAM001246985'
83.11
-50.09
2
0
4.94
0
1.35
0.06
'7746693'
357.76
0
5
5
82.69
-31.5
0
0
8.63
0
1.55
2.53
'SAM001246635'
82.55
-23.82
0.25
0
9.98
0
0.94
0.01
'5107893'
565.03
1
6
3.8
82.37
-63.75
2.99
0
2
0
1.4
0.46
'SAM001246618'
81.88
-46.11
0.27
0
6
0
0.63
0.11
'SAM001246695'
80.84
-68.24
1
0
2
0
1.25
0.11
'SAM001246863'
80.62
-30.59
1.78
0
8.98
6.44
1.56
0.32
'SAM001246806'
80.56
-54.58
0.85
0
3.99
0
1.82
3.15
'SAM001246602'
80.11
-42.34
0.38
0.84
6
0.09
1.91
1.99
'SAM001246625'
79.71
-40.64
1.32
0
5.99
0.11
0.39
0.06
'5282931'
316.48
3
4
5.1
79.48
-41.19
3.01
0
4.94
0
1.46
2.56
'SAM001246818'
78.85
-23.92
5.67
0.95
5.99
0
0.46
0.03
'5175515'
367.25
0
3
4.8
78.85
-18.56
1.58
0
9.96
0
2.23
0.13
'5192853'
333.45
0
4
2.5
78.7
-49.7
4.87
0
2.94
0
2.06
0.66
'SAM001246560'
78.6
-45.26
6.61
0
3
0
2.51
0.2
'SAM001246852'
77.84
-40.82
1.06
0
5.94
0
0.9
0.02
'SAM001246593'
76.55
-36.98
1.68
0
5.97
0
0.66
0.01
'5101823'
376.81
0
4
3.3
76.44
-60.65
1
0
2.84
0
2.37
0.35
'SAM001247090'
76.16
-42.25
1.76
0
4.98
0
0.63
0.01
'7591231'
385.46
3
6
0.4
76.07
-60.72
1.58
0
2.21
0
1.84
0.91
'SAM001247084'
76.03
-77.09
1
0
0
0
4.48
4.9
'SAM001246793'
75.83
-32.33
2.98
0
6
0.34
2.07
3.06
'SAM001246530'
75.7
-50.56
0.85
0
3.99
0
0.83
0.25
'7796224'
395.43
1
5
3.8
75.23
-30.61
4.84
0
6
3.28
1.38
0.13
'7726180'
384.81
1
6
2.5
75.05
-59.23
1.37
0
2
0
0.98
0.53
'SEW06312SC'
384.74
1
7
5.1
74.74
-37.28
2.86
0
4.98
0.11
0.47
0.02
'7437091'
311.4
2
2
2.7
74.64
-65.04
1.3
0
2
3.67
2.86
3.08
'SPB02803SC'
433.41
1
9
4.1
74.43
-32.5
2.2
0
6
0
0.42
0.04
'6407567'
363.45
1
4
4.6
74.3
-42.62
1
0
4.97
0
0.61
0.07
'7724431'
366.41
0
5
4.5
74.23
-19.12
2.94
0
7.93
0
0.64
0.01
6433056'
72.81
-33.01
1.7
0
5.94
0
1.5
2.08
'7676009'
72.78
-55.06
0.89
0
2.84
0
1.02
0.05
'6703891'
758.04
3
7
10.1
72.71
-32.07
1.88
0
6
0
0.52
0.03
'7842136'
72.54
-40.23
0
0
5.61
0
1.89
2.45
'HTS09290SC'
436.29
1
5
6.4
72.44
-48.11
0.9
0
3.9
0
1.14
0.37
'7752888'
412.4
0
8
3.7
71.42
-34.02
0.79
0
6
0
0.49
0.03
'7397377'
70.89
-37.94
1.63
0
4.94
0
0.8
0.02
'7761278'
70.81
-57.38
1.1
0
2
0
1.33
0.78
'SPB01807SC'
501.94
3
9
4.6
70.72
-31.67
3.32
0
4.98
0
0.71
0.62
'5256974'
70.41
-42.9
2.46
0
3.7
0
1.22
0.27
'7706580'
393.5
1
6
3.8
70.2
-29.58
1.74
0
5.99
0
1.41
2.25
'6629394'
70.17
-34.44
0
0
6
0
0.15
0.03
'5490779'
70.17
-49.67
2.18
0.53
2.43
0
2.31
2.41
'HTS01560SC'
479.82
2
10
3
70.13
-30.04
1.64
0
5.88
0
0.06
0
'7814264'
70.01
-44.42
4.61
0
2
0
0.13
0.01
'7679150'
69.48
-34.72
0
0
6
0
0.64
0.04
'6565654'
69.48
-28.04
2
0
5.96
0
0.18
0.02
'7907769'
69
-54.93
0.69
0.44
1.99
0
0.7
0.11
'7785278'
283.32
1
3
3.3
68.97
-38.96
0.75
0
4.93
0
0.95
0.01
'7589277'
68.9
-30.61
1
0
5.97
0
0.28
0.01
'5135469'
68.84
-41.39
0
0
4.91
0
1.03
0.04
'7928256'
68.37
-50.64
2.25
0
2
0
2.2
3.34
'SPB02121SC'
527.23
2
7
5.4
67.99
-27.32
1.62
0
6
0
0.1
0.01
'9040855'
67.96
-49.19
0.96
0
2.91
0
0.8
0.03
'9027311'
67.91
-45.37
2.01
0
3
0
0.76
0.06
'7957875'
67.86
-33.79
0.03
0
6
0
1.02
0.01
'7278181'
67.5
-48.68
1.04
0
2.88
0
0.89
0.14
'JFD02812SC'
67.09
-42.38
2.58
0
2.99
0
1.58
2.23
'SCR01399SC'
307.37
1
5
1.2
67.04
-40.04
3.43
0
3
0
1.63
1.97
'7803112'
369.14
1
5
1.7
66.9
-38.5
2.23
0
3.97
0
1.1
0.07
'7186658'
394.42
1
6
3.6
65.89
-39.87
2.03
0
3.73
0
1.33
0.21
'BTB08339SC'
452.29
1
8
4.7
65.3
-59.22
0.98
0
0.94
0
3.66
4.45
'BTB05187SC'
64.94
-35.92
4.36
0
2.99
0
2.08
2.12
'BTB08116SC'
283.23
2
8
-0.1
64.89
-48.24
0
0
2.94
0
0.51
0.03
'HTS11122SC'
324.36
0
7
3.2
64.67
-45.05
3.07
0
2
0
0.83
0.08
'5153298'
210.23
1
4
1.9
64.47
-54.36
1.95
0
0.94
0
0.76
0.07
'GK01883SC'
64.43
0.27
1.75
0
9.91
0
0.03
0.03
'RH00719SC'
64.41
-39.29
0.75
2.4
2.67
0.07
0.42
0.51
'KM09475SC'
64.4
-42.42
1
0.43
3
0
0.29
0.3
'5380289'
265.39
2
2
3.7
64.24
-39.11
0.95
0
3.92
0.06
0.62
0.06
'KM10142SC'
63.41
-55.23
1.67
0
0.99
0
1.57
0.4
'NRB00168SC'
63.32
-56.42
0.68
0
1
0
0.73
0.31
'7833474'
62.78
-41.42
0
0
3.91
1.05
0.66
0.26
'S14452SC'
62.54
-50.79
0.99
0
1.99
0
1.67
0.21
'7646744'
62.4
-47.49
1.59
0
1.97
0
0.86
0.01
'7811263'
62.38
-46.17
0.54
0
3
0.01
2.73
2.05
'DFP00065SC'
62.38
-42.44
2.94
0
1.96
0
0.41
0.16
'SPB03042SC'
62.2
-30.09
1.16
0
4.89
0
0.36
0
'SPB01985SC'
61.78
-48.5
0.69
0
2
0.11
0.6
0.52
'RH01386SC'
61.22
-53.84
0.93
0
1
0
0.78
0.06
'7637680'
60.47
-35.35
3.39
0
2.99
0.08
1.65
0.42
'5587264'
60.31
-40.27
2.94
0
2
0
0.49
0.2
'7748642'
60.27
-37.49
2.29
0
3
0
1.12
0.18
'BTB08347SC'
59.74
-43.1
1.94
0
1.99
0.09
0.55
0.1
'5470088'
59.62
-51.13
1.37
0
1
0.18
1.22
1
'HTS00581SC'
59.42
-42.55
2.63
0
1.94
0
1.38
0.12
'7733855'
59.25
-41.32
2.55
0
1.98
0.92
2.93
5.08
'SEW06627SC'
59.01
-27.54
2.87
0
3.87
0
0.17
0.01
'RJF00786SC'
59
-44.15
1.46
0
1.94
0
0.66
0.05
'5915863'
58.85
-41.4
1.9
0
2.46
0
2.81
2.63
'SEW02685SC'
58.68
-43.99
1.59
2
1
0
1.04
0.01
'SCR00533SC'
58.44
-47.18
0
0
2
0
0.45
0.06
'SPB02109SC'
58.38
-36.89
1.88
0
3
0
2.18
2.25
'HTS02479SC'
58.04
-41.6
2
0
2
0
0.81
0.07
'5153298'
57.6
-46.88
1.57
0.76
1
0
1.2
0.15
'6060460'
57.42
-59.34
0
0
0
0
3.07
4.21
'5115900'
57.26
-23.84
3.44
0
3.97
0
0.45
0.2
'5584856'
55.63
-31.1
2.37
0
2.99
0
1.24
1.98
'5648649'
54.82
-38.2
1.69
0
2
0
1.23
2.02
'5852635'
54.47
-53.89
0.99
0
0
0
1.38
0.37
'7477596'
54.2
-36.08
2.73
0
2
0
1.13
0.16
'5753084'
53.94
-43.47
0.67
0
1.69
0
1.03
0.22
'5753084'
53.9
-40.29
3.44
0
0.94
0
1.28
0.11
'5153298'
53.79
-36.72
1.7
0
2
0
1.02
2.01
'5852635'
Week 11 & 12 Figure 1: SDS-Page gel for Trial 2 of protein purification for P. vivax STPP 2B.
Lane 1: ColorPlus Prestained Protein Ladder
Lane 2: Cell lysate after induction
Lane 3: Soluble Fraction
Lane 4: Flow through
Lane 5: Wash
Lane 6: Elution 1
Lane 7: Elution 2
Analysis:
The protein is visible in the cell lysate after induction. However, it is once again lost during the purification process. There is a possibility that the protein was not in the soluble fraction. The protein has been expressed again and will be suspended in a sonication buffer that includes glycerol and Triton X in order to help solubilize the protein. If the protein is not present in the soluble fraction, it is a membrane protein and can not be purified using our methods.
Table 1: Top 40 ligands from virtual screening of HF9PlatesPlates5_9 vs P. vivax STPP 2B.
Score
S(PLP)
S(hbond)
S(cho)
S(metal)
DE(clash)
DE(tors)
intcor
time
Ligand name
75.05
-59.23
1.37
0
2
0
0.98
0.53
5.052
'SEW06312SC'
74.64
-65.04
1.3
0
2
3.67
2.86
3.08
5.468
'SPB02803SC'
72.54
-40.23
0
0
5.61
0
1.89
2.45
4.616
'HTS09290SC'
70.81
-57.38
1.1
0
2
0
1.33
0.78
6.394
'SPB01807SC'
70.17
-49.67
2.18
0.53
2.43
0
2.31
2.41
6.108
'HTS01560SC'
68.37
-50.64
2.25
0
2
0
2.2
3.34
5.703
'SPB02121SC'
67.5
-48.68
1.04
0
2.88
0
0.89
0.14
2.457
'JFD02812SC'
67.09
-42.38
2.58
0
2.99
0
1.58
2.23
3.302
'SCR01399SC'
65.89
-39.87
2.03
0
3.73
0
1.33
0.21
4.649
'BTB08339SC'
65.3
-59.22
0.98
0
0.94
0
3.66
4.45
5.814
'BTB05187SC'
64.94
-35.92
4.36
0
2.99
0
2.08
2.12
4.107
'BTB08116SC'
64.89
-48.24
0
0
2.94
0
0.51
0.03
3.243
'HTS11122SC'
64.47
-54.36
1.95
0
0.94
0
0.76
0.07
2.41
'GK01883SC'
64.43
0.27
1.75
0
9.91
0
0.03
0.03
0.62
'RH00719SC'
64.41
-39.29
0.75
2.4
2.67
0.07
0.42
0.51
3.767
'KM09475SC'
64.24
-39.11
0.95
0
3.92
0.06
0.62
0.06
2.802
'KM10142SC'
63.41
-55.23
1.67
0
0.99
0
1.57
0.4
6.115
'NRB00168SC'
62.78
-41.42
0
0
3.91
1.05
0.66
0.26
1.821
'S14452SC'
62.38
-46.17
0.54
0
3
0.01
2.73
2.05
9.214
'DFP00065SC'
62.38
-42.44
2.94
0
1.96
0
0.41
0.16
5.199
'SPB03042SC'
62.2
-30.09
1.16
0
4.89
0
0.36
0
2.527
'SPB01985SC'
61.78
-48.5
0.69
0
2
0.11
0.6
0.52
4.87
'RH01386SC'
60.27
-37.49
2.29
0
3
0
1.12
0.18
3.992
'BTB08347SC'
59.62
-51.13
1.37
0
1
0.18
1.22
1
8.412
'HTS00581SC'
59.25
-41.32
2.55
0
1.98
0.92
2.93
5.08
5.064
'SEW06627SC'
59.01
-27.54
2.87
0
3.87
0
0.17
0.01
3.29
'RJF00786SC'
58.85
-41.4
1.9
0
2.46
0
2.81
2.63
6.749
'SEW02685SC'
58.68
-43.99
1.59
2
1
0
1.04
0.01
2.819
'SCR00533SC'
58.44
-47.18
0
0
2
0
0.45
0.06
3.529
'SPB02109SC'
58.38
-36.89
1.88
0
3
0
2.18
2.25
3.606
'HTS02479SC'
57.96
-48.95
1.96
0
0.94
1.02
1.93
2.29
4.921
'HTS09507SC'
57.85
-38.21
1.41
0
2.9
0.68
1.74
2.19
2.836
'KM01046SC'
57.77
-38.18
1.13
0
3
0
2.41
3.03
4.642
'HTS03338SC'
57.37
-50.63
0.99
0
1
0
1.32
0.38
7.171
'BR00114SC'
57.03
-40.94
1.88
0
1.97
0
1.35
1.25
6.062
'CD03598SC'
56.8
-41.36
0.36
0
2.84
0
3.61
4.54
5.649
'BTBG00099SC'
56.74
-49.37
0.98
0.24
0.97
0.16
1.15
0.37
4.844
'HTS08498SC'
56.6
-42.17
1
0
1.99
0
0.34
0.14
2.594
'SEW00880SC'
56.33
-48.5
0
0
1.94
0
2.11
0.39
0.829
'SCR00407SC'
56.1
-36.17
1.49
0
2.94
0
1.23
0.11
1.166
'KM07666SC'
56.07
-51.17
0.19
0
1
0
3.76
5.83
4.62
'KM09000SC'
Analysis
The scores for the top ranking ligands are relatively high. Lipinski's rule data should be added to the top ligands in order to narrow down which ligands will be good for use in inhibition assays.
Week 9 & 10
VIRTUAL DATA:
Homology model template: 1M63 (chain A)
Figure 1: Homology model for P. vivax STPP as surface with carbon in green, nitrogen in blue, oxygen in red. Zinc ion as gray sphere and Fe ion as orange sphere. Cyclopentadiene as sticks with carbon in pink, hydrogen in gray/
Cyclopentadiene was added to the active site of the homology model in order to be able to define the active site in Gold. In Gold, the three histidine residues in the active site were deprotonated and one was flipped in order to have the lone pair pointing toward the positively charged metal ions.
Table 1: Positive control ligands with data for Lipinski's rule. Ligands were found using BindingDB, TDRtargets, and literature.
Name
Mol. Weight
H don
H acc
LogP
From
10366183 (Bos taurus)
444.56
3
5
3.6
BindingDB
44372867 (Homo sapiens
466.61
2
7
2.5
BindingDB
pNPP (378)
219.09
2
6
0.3
PubChem
5944
196.19
0
4
0.6
TDRTargets
44372805
594.69
5
11
0.3
BindingDB
44330406 (Bos taurus)
430.54
3
5
3.2
BindingDB
44330465 (Bos taurus)
400.51
3
4
3.9
BindingDB
44330390 (Bos taurus)
514.6
3
7
3.1
BindingDB
44330422 (Bos taurus)
514.6
3
7
3.1
BindingDB
93004
168.15
0
4
-0.1
TDRTargets
Table 2: Negative control ligands found using the Chembridge database.
Name
Mol. Weight
H don
H acc
LogP
From
'44330422'
514.61
3
7
3.1
Chembridge
'44372805'
594.69
5
11
0.3
Chembridge
'2244_aspirin'
180.157
1
4
1.2
PubChem
'6338765'
339.4
1
4
2.9
Chembridge
'171044'
445.037
2
3
7.4
Chembridge
'3851230'
505.76
3
6
4.7
Chembridge
'6808645'
376.47
2
6
2.7
Chembridge
Table 3: Control docking results for Control Library against P. vivax STPP homology model.
Score
S(PLP)
S(hbond)
S(cho)
S(metal)
DE(clash)
DE(tors)
intcor
time
Ligand name
90.85
-48.7
3.06
0
5.94
0
1.92
0.85
117.033
'44330406'
88.7
-60.46
2.81
0
3.88
0
3
1.66
151.751
'44330390'
74.66
-35.92
1.89
0
5.99
0
1.77
0.67
120.668
'10366183'
68.78
-13.18
2.17
0
8.93
0
2.38
0.26
160.067
'44372805'
67.98
-52.5
1.74
0
2
0
1.27
0.62
103.366
'44330465'
67.68
-12.83
2.61
0
7.94
0
0.3
0
9.694
'378'
67.65
-54.42
2.02
0
2
0.46
3.03
1.01
146.628
'44330422'
66.07
-46.24
4.35
0
1.97
0
3.3
1.55
156.914
'44330422'
62.27
-50.38
4.29
0
0
0
0.69
0.24
158.462
'44372805'
60.6
-19.02
2.05
0
6
0
0.28
0.01
8.043
'2244_aspirin'
59.89
-56.43
1.98
0
0
0
2.24
2
142.827
'44330390'
52.3
-43.1
0.01
0
1.88
0
1.6
1.08
39.562
'6338765'
51.83
-46.58
0.46
0
0.94
0
1.07
0.36
70.104
'44372867'
51.75
-47.25
2.16
0
0
0
1.26
0.29
96.266
'171044'
51.56
-37.57
1.59
0
2
0
1.79
0.81
74.206
'3851230'
50.58
-49.25
1.22
0
0
0
1.35
0.36
68.347
'44372867'
50.26
-45.89
1.99
0
0
0
0.98
0.37
75.627
'44372867'
43.54
-42.82
0.62
0
0
0
1.57
1.96
23.012
'6808645'
42.64
-42.91
0.4
0
0
0
1.83
2.07
21.389
'6808645'
33.83
-17.33
1.56
0
1.97
0
0
0
2.491
'93004'
30.1
-25.8
1.43
0
0
0
0
0
6.077
'5944'
Analysis: The top-ranking ligands in the control run were from the positive control ligands that were known to bind to STPP in other organisms. This indicates that using this homology model will give relatively accurate outputs. Aspirin (a negative control) was ranking above some positive control ligands. The best ranking poses for these ligands will be analyzed in PyMol.
WET LAB DATA:
Protein purification round 1:
1 500 mL large culture was grown up.
Pellet weight: 3.29 g
Figure 2: Elution 1 of P. vivax STPP 2b. Protein was eluted into 2 mL of elution buffer. Conc. was calculated using molar extinction coefficient specific to the protein. Conc.= 0.62 mg/mL. Total amount of protein= 1.25 mg.
Figure 3: Elution 2 of P. vivax STPP 2b. Remaining protein was eluted into 4 mL of elution buffer. Conc. was calculated using specific molar extinction coefficient. Conc= 0.31 mg/mL. Total amount of protein= 1.25 mg.
Figure 4: SDS-Page gel for P. vivax STPP purification
Lane 1: ColorPlus Prestained Protein Ladder
Lane 2: Cell lysate after induction
Lane 3: Soluble fraction
Lane 4: Flow through
Lane 5: Wash
Lane 6: Elution 1
Lane 7: Elution 2
ColorPlus Prestained Protein Ladder:
Analysis: Elution 1 should have had a higher protein concentration than did Elution 2. This indicates that there may have been problems with purification (e.g. contamination). In the SDS-Page gel, it can be seen that the protein (63 kDa) was expressed well after induction. However, it was lost during the purification process. Possible explanations are potential insoluble portions of the protein or protein aggregation that hides the His6 tag and prevents proper binding to the Ni-NTA column.
Protein purification round 2:
2 500 mL large cultures were grown up.
Pellet weight:
Culture 1: 5.42 g
Culture 2: 3.82 g
Only culture 1 has been purified using the same purification method as before. Purification buffers (wash and elution) were made again and pHed.
Figure 5: Elution 1 of P. vivax STPP 2b. Protein was eluted into 5 mL of elution buffer. Conc. was calculated using molar extinction coefficient specific to the protein. Conc.= 0.42 mg/mL. Total amount of protein= 2.08 mg.
Figure 6: Elution 2 of P. vivax STPP 2b. Remaining protein was eluted into 4 mL of elution buffer. Conc. was calculated using specific molar extinction coefficient. Conc= 0.1 mg/mL. Total amount of protein= 0.4 mg.
Analysis: Elution 1 seems to have a much larger amount of protein than Elution 2, which is a good indication that protein purification was more successful than the first time. An SDS-Page gel will have to be run in order to analyze whether the protein is present in Elution 1 and if it is pure. ------------------------------------------------------------------------------------------------------------------------------------------------------------ Week 7 & 8
Nice work. Good captions and analysis.Do you have any virtual data? Thank you. -Max 10/21/13
It was confirmed that colony 2 on the master plate was a positive clone.
Figure 1: Concentration of Colony #2 mini-prepped plasmid P. vivax STPP in pNIC-Bsa4 vector.
Colony #2 Nucleotide Blast with DNA sequencing results (pLIC-Forward primer)
Sequence ID: lcl|235163Length: 1292Number of Matches: 1 Related Information Range 1: 101 to 1083Graphics Next Match Previous Match
The remaining mini-prepped sample of STPP in pNIC-Bsa4 from colony 2 was used to transform E. coli BL21(DE3) bacteria in order to prepare for protein expression.
Figure 2: Control plate. LB Agar+Kan+Sucrose plate with untransformed E. coli BL21(DE3) bacteria.
There are no colonies on the control plate. This indicates that sterile technique was good because the Kanamycin (an antibiotic) killed the untransformed cells because they lack Kanamycin resistance.
Figure 3: BL21(DE3) transformed with P. vivax STPP grown on LB+Kan+Suc plates. 10 uL of the final transformation mixture was plated.
There are very few colonies on this plate, which indicates that 10 uL may have been too small an amount to plate for optimal growth of the bacteria.
Figure 4: BL21(DE3) transformed with P. vivax STPP grown on LB+Kan+Suc plates. 50 uL of the final transformation mixture was plated.
There are many more colonies on this plate than were on the 10 uL plate. This indicates that adding 50 uL of the transformation mixture was much more desirable for bacterial growth.
More DH5-Alpha with STPP in pNIC-Bsa4 from colony #2 on the master plate were grown up in an overnight culture, spun down, and midi-prepped.
Figure 5: Concentration of midi-prepped P. vivax STPP in pNIC-Bsa4. Conc= 68.4 ng/uL.
This midi-prepped plasmid can be used in the future to transform more BL21(DE3) as needed. Midi-prepping is cleaner than mini-prepping, so this sample is likely cleaner than the mini-prepped sample used for the first round of transformation of BL21(DE3).
Figure 6: Concentration of purified P. vivax STPP Elution 1. Conc= 0.46 mg/mL.
FIgure 7: Concentration of purified P. vivax STPP Elution 2. Conc= 0.23 mg/mL.
Summary/Analysis:
Elution 1 of the purified protein is more highly concentrated than Elution 2, which is to be expected. Elution 2 is the remained of the protein that is eluted for a second time from the Ni-NTA resin. Samples were taken throughout the purification process and SDS-Page gel will be run to characterize the protein and determine its purity. If protein is impure, FPLC will be done.
Week 5 &6
Nice work. Try to include some analysis of your data. Thank you. -Max 10/07/2013
Another attempt was made to grow up DH5-alpha with pNIC-Bsa4. Again, the yield was very small. Transformation of DH5-alpha cells with the pNIC-Bsa4 plasmid was done again in order to get new colonies from which to grow the bacteria.
Figure 1: Nanodrop reading for midi-prepped pNIC-Bsa4 plasmid. Concentration is 55.3 ng/uL.
Figure 2: Nanodrop reading (Trial 1) of cut plasmid pNIC-Bsa4. Concentration is 8.2 ng/uL.
The concentration of the cut plasmid is very low.
Figure 3: Nanodrop reading (Trial 2) of cut and PCR cleaned plasmid pNIC-Bsa4. Concentration is 151.8 ng/uL.
Three tubes were prepared, each with total volume of 50 uL. All samples were mixed into one tube during PCR cleanup, and the plasmid was eluted into 30 uL of buffer.
Figure 4: Plasmodium vivax serine-threonine phosphatase, catalytic subunit 2B gene in plasmid vector pNIC-Bsa4 in DH5-Alpha cells. Plates are LB+Kan+Sucrose. Cloned with 2 uL accepting vector (pNIC-Bsa4) and 4 uL gene insert.
Figure 5: Plasmodium vivax serine-threonine phosphatase, catalytic subunit 2B gene in plasmid vector pNIC-Bsa4 in DH5-Apha cells. Plates are LB+Kan+Sucrose. Cloned with 2 uL accepting vector (pNIC-Bsa4) and 8 uL gene insert.
Figure 6: Master plate for Plasmodium vivax serine-threonine phosphatase, cataytic subunit 2B gene in pNIC-Bsa4 plasmid vector in DH5-Alpha cells. Plates are LB+Kan+Sucrose.
Summary/analysis:
The first cloning attempt was made for this target. Bacterial colonies were present on the LB+Kan+Sucrose plates for both the original plate and the master plate, which indicates that the SacB gene is not in these bacteria. Mini-prep, RE digest, and sequencing will be performed next week in order to determine if these are positive clones.
Week 3 & 4 Figure 1: Secondary PCR for Plasmodium vivax serine-threonine phosphatase, catalytic subunit 2B, putative gene. Lane 1: 1 kb ladder Lane 5: Secondary PCR (using primary PCR as template) Lanes 2-4 were PCR samples of another researcher.
Analysis: Lane 5 does not show a distinct band, so the secondary PCR was unsuccessful.
Figure 2: Secondary PCR (Trial 2) of Plasmodium vivax serine-threonine phosphatase, catalytic subunit 2B, putative gene. Lane 1: 1 kb ladder Lane 2: empty Lane 3: Secondary PCR (using primary PCR as template)
Analysis: Lane 3 shows a distinct band between 1.5 and 2.0 kilobases. The target gene is 1,977 bp, so the PCR seems to be successful. For this PCR, the annealing temperature used was 59 degrees C, and the time for annealing and elongation was increased to 30 seconds each.
Lane 1: 1 kb ladder Lane 2: Secondary PCR (Ariel C.) Lane 3-6: PCR2 reaction with identical amounts of reagents
Analysis: Lanes 3-6 show very distinct bands between 1.5 and 2.0 kb. Because the P. vivax STPP gene is 1,977 bp, it can be concluded that the PCR was successful in amplifying the gene. Annealing temperature: 59 degrees.
Figure 4: Nanodrop reading after PCR cleanup on PCR2 product for Plasmodium vivax serine-threonine phosphatase, catalytic subunit 2B, putative gene.
Analysis: The 260/280 and 260/230 values are very close to 2, which indicates that the DNA sample is pure.
Figure 5: Nanodrop reading of pNIC-Bsa4 plasmid (from DH5-alpha) after midi-prep.
Analysis: The nano drop reading shows a negative yield for the plasmid, which indicates that the plasmid was not in the midi-prepped sample. The cell pellet from the overnight culture of DH5-alpha was very small (even after several trials), which indicates that there is a problem with the plate. Another overnight culture will be prepared for midi-prep.
Summary: Secondary PCR and PCR^2 reactions were completed for P. vivax serine-threonine phosphate, catalytic subunit 2B, putative. A Nanodrop reading was taken and the sample was found to be relatively pure. Cells of DH5-alpha transformed with the pNIC-Bsa4 plasmid were midi-prepped, but there was no plasmid yield. The next step is to grow more overnight culture of DH5-alpha and perform midi-prep again. Week 1 & 2 Figure 1: Primary PCR for Plasmodium vivax serine-threonine phosphatase, catalytic subunit 2B, putative.
Lane 1: 1 kb ladder
Lane 9: Primary PCR (PCR on oligo mix for P. vivax STPP 2B)
Lanes 2-8 were PCR samples of other researchers.
Summary:
This week I made an oligo mix for the primers that will form the gene which codes for P. vivax serine-threonine phosphatase, catalytic subunit 2B, putative. I then did a primary PCR with the oligo mix.
I grew an overnight culture of pNIC-Bsa4 in DH5-Alpha. The cells have been spun down in a centrifuge. These will be midi-prepped in the future.
Analysis:
The "smear" that appears on Lane 9 of the gel indicates that the primary PCR was successful. The smear should be composed of several separate strands of DNA that will be put together with secondary PCR.
Fall 2013
WEEK 4 Figure 1: RE digest of pGBR22 using Restriction enzymes EcoRI-HF and PvuII (left side of gel)
Lane 1: empty
Lane 2: 1 kb ladder
Lane 3: Uncut plasmid
Lane 4: pGBR22 with EcoRI-HF in NE buffer 2
Lane 5: pGBR22 with PvuII in NE buffer 2
Lane 6: pGBR22 with PvuII and EcoRI-HF in NE buffer 2
Lane 7: pGBR22 with EcoRI-HF in NE buffer for EcoRI
Absorbance measurement of yopH Elution 1 at 280 nm. Absorbance is 0.940.
Absorbance measurement of yopH Elution 2 at 280 nm. Absorbance is 0.109. --------------------------------------------------------------------------------------------------------------------------------------------------------------------------- WEEK 3 Figure 1: Trial 1 PCR of pGFP. DNA failed to amplify
Lane 1: 1 kb ladder
Lane 2: Sample A, VDS primers
Lane 3: Sample B, VDS primers
Lane 4: Sample C, VDS primers
Lane 5: 1 kb ladder
Lane 6: Sample D, VDS primers
Lane 7: Sample E, M13 primers
Lane 8: Sample F: M13 primers
Lane 9: Sample G, M13 primers
Lane 10: Sample H: M13 primers Figure 1: Trial 2 PCR of pGFP. DNA may have amplified in lane 4, but PCR needs to be redone with 100 bp ladder.
Lane 1: 1 kb ladder
Lane 2: Sample A, VDS primers
Lane 3: Sample B, VDS primers
Lane 4: Sample C, VDS primers
Lane 5: Sample D, VDS primers
Lane 6: 1 kb ladder
Lane 7: Sample E, M13 primers
Lane 8: Sample F: M13 primers
Lane 9: Sample G, M13 primers
Lane 10: Sample H: M13 primers ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------- WEEK 2
Figure 1: PCR of plasmid pGBR22 (left side of gel).
Anita Vasudevan 6/13/2013 VDS
PCR of plasmid: pGBR22
Lane 1: 1 kb Ladder
Lane 2: Sample A, 1:500 template DNA, 1 uL Taq
Lane 3: Sample B: 1:500 template DNA, 1 uL Taq
Lane 4: Sample C: 1:50 template DNA, 1 uL Taq
Lane 5: Sample D: 1:50 template DNA, 1 uL Taq
Figure 2: Nanodrop spectrometer Run 1 reading for puc19 plasmid purified from E.coli (DH5-alpha). Concentration is 49.0 ng/uL
Figure 3: Nanodrop spectrometer Run 2 reading for puc19 plasmid purified from E. coli (DH5-alpha). Concentration is 41.4 ng/uL WEEK 1
Figure 1: E. coli (DH5-alpha) cells transformed with 1 ng puc19 plasmid grown on LB+Amp Agar plates.
To count colonies:
~divided into 45 equal squares
~approx. 28 colonies per square
~(45*28)= 1,260 colonies
Transformation efficiency: 1,260 colonies/1 ng= 1,260 colonies/ng
Figure 2: E. coli (DH5-alpha) cells transformed with 5 ng puc19 plasmid grown on LB+Amp Agar plates.
To count colonies:
~divided into 45 equal squares
~approx. 67 colonies per square
~(45*67)= 3,015 colonies
Transformation efficiency: 3,015 colonies/5 ng= 603 colonies/ng
Figure 3: E. coli cells (DH5-alpha) transformed with 25 ng puc19 plasmid grown on LB+Amp Agar plates.
To count colonies:
~divided into 45 equal squares
~approx. 12 colonies per square
~(45*12)= 540 colonies
Table 1: Top 31 ligands from virtual screening of CB306 vs P. vivax STPP 2B.
Table 2: Top 45 ligands from virtual screening of NIHClinicalCollection vs P. vivax STPP 2B.
Table 4: Best ranking list for all libraries screened against P. vivax STPP 2B homology model.
Week 11 & 12
Figure 1: SDS-Page gel for Trial 2 of protein purification for P. vivax STPP 2B.
Lane 1: ColorPlus Prestained Protein Ladder
Lane 2: Cell lysate after induction
Lane 3: Soluble Fraction
Lane 4: Flow through
Lane 5: Wash
Lane 6: Elution 1
Lane 7: Elution 2
Analysis:
The protein is visible in the cell lysate after induction. However, it is once again lost during the purification process. There is a possibility that the protein was not in the soluble fraction. The protein has been expressed again and will be suspended in a sonication buffer that includes glycerol and Triton X in order to help solubilize the protein. If the protein is not present in the soluble fraction, it is a membrane protein and can not be purified using our methods.
Table 1: Top 40 ligands from virtual screening of HF9PlatesPlates5_9 vs P. vivax STPP 2B.
Analysis
The scores for the top ranking ligands are relatively high. Lipinski's rule data should be added to the top ligands in order to narrow down which ligands will be good for use in inhibition assays.
Week 9 & 10
VIRTUAL DATA:
Homology model template: 1M63 (chain A)
Figure 1: Homology model for P. vivax STPP as surface with carbon in green, nitrogen in blue, oxygen in red. Zinc ion as gray sphere and Fe ion as orange sphere. Cyclopentadiene as sticks with carbon in pink, hydrogen in gray/
Cyclopentadiene was added to the active site of the homology model in order to be able to define the active site in Gold. In Gold, the three histidine residues in the active site were deprotonated and one was flipped in order to have the lone pair pointing toward the positively charged metal ions.
Table 1: Positive control ligands with data for Lipinski's rule. Ligands were found using BindingDB, TDRtargets, and literature.
Table 2: Negative control ligands found using the Chembridge database.
Table 3: Control docking results for Control Library against P. vivax STPP homology model.
Analysis: The top-ranking ligands in the control run were from the positive control ligands that were known to bind to STPP in other organisms. This indicates that using this homology model will give relatively accurate outputs. Aspirin (a negative control) was ranking above some positive control ligands. The best ranking poses for these ligands will be analyzed in PyMol.
WET LAB DATA:
Protein purification round 1:
1 500 mL large culture was grown up.
Pellet weight: 3.29 g
Figure 2: Elution 1 of P. vivax STPP 2b. Protein was eluted into 2 mL of elution buffer. Conc. was calculated using molar extinction coefficient specific to the protein. Conc.= 0.62 mg/mL. Total amount of protein= 1.25 mg.
Figure 3: Elution 2 of P. vivax STPP 2b. Remaining protein was eluted into 4 mL of elution buffer. Conc. was calculated using specific molar extinction coefficient. Conc= 0.31 mg/mL. Total amount of protein= 1.25 mg.
Figure 4: SDS-Page gel for P. vivax STPP purification
Lane 1: ColorPlus Prestained Protein Ladder
Lane 2: Cell lysate after induction
Lane 3: Soluble fraction
Lane 4: Flow through
Lane 5: Wash
Lane 6: Elution 1
Lane 7: Elution 2
ColorPlus Prestained Protein Ladder:
Analysis: Elution 1 should have had a higher protein concentration than did Elution 2. This indicates that there may have been problems with purification (e.g. contamination). In the SDS-Page gel, it can be seen that the protein (63 kDa) was expressed well after induction. However, it was lost during the purification process. Possible explanations are potential insoluble portions of the protein or protein aggregation that hides the His6 tag and prevents proper binding to the Ni-NTA column.
Protein purification round 2:
2 500 mL large cultures were grown up.
Pellet weight:
Culture 1: 5.42 g
Culture 2: 3.82 g
Only culture 1 has been purified using the same purification method as before. Purification buffers (wash and elution) were made again and pHed.
Figure 5: Elution 1 of P. vivax STPP 2b. Protein was eluted into 5 mL of elution buffer. Conc. was calculated using molar extinction coefficient specific to the protein. Conc.= 0.42 mg/mL. Total amount of protein= 2.08 mg.
Figure 6: Elution 2 of P. vivax STPP 2b. Remaining protein was eluted into 4 mL of elution buffer. Conc. was calculated using specific molar extinction coefficient. Conc= 0.1 mg/mL. Total amount of protein= 0.4 mg.
Analysis: Elution 1 seems to have a much larger amount of protein than Elution 2, which is a good indication that protein purification was more successful than the first time. An SDS-Page gel will have to be run in order to analyze whether the protein is present in Elution 1 and if it is pure.
------------------------------------------------------------------------------------------------------------------------------------------------------------
Week 7 & 8
Nice work. Good captions and analysis.Do you have any virtual data? Thank you. -Max 10/21/13
It was confirmed that colony 2 on the master plate was a positive clone.
Figure 1: Concentration of Colony #2 mini-prepped plasmid P. vivax STPP in pNIC-Bsa4 vector.
Colony #2 Nucleotide Blast with DNA sequencing results (pLIC-Forward primer)
Sequence ID: lcl|235163Length: 1292Number of Matches: 1
Related Information
Range 1: 101 to 1083Graphics Next Match Previous Match
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 101 ATGGAACCGCTGCCGAACCCGAAAAACGACCGTCAGGTCAAAGATGTTGAGCCGCCACCG 160
Query 61 GCGAAACCACTGAGCCTGGAACTCCTGTACCCGAATGGTACCGATGAACCGCCTGATTAC 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 161 GCGAAACCACTGAGCCTGGAACTCCTGTACCCGAATGGTACCGATGAACCGCCTGATTAC 220
Query 121 AAGGCGCTGCGTGACCATCTGAAAAAAGAAGGTCGCATCCGCAAGGAGGATTGCCTGGAC 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 221 AAGGCGCTGCGTGACCATCTGAAAAAAGAAGGTCGCATCCGCAAGGAGGATTGCCTGGAC 280
Query 181 ATCATCAAACGTGTAATCGACATCGTTAGCAACGAACCGAACCTGCTGCGTCTGCAAGAC 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 281 ATCATCAAACGTGTAATCGACATCGTTAGCAACGAACCGAACCTGCTGCGTCTGCAAGAC 340
Query 241 CCGATCACTATCGTTGGTGACATTCATGGTCAGTACTACGACTTCCTGAAACTGCTGGAA 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 341 CCGATCACTATCGTTGGTGACATTCATGGTCAGTACTACGACTTCCTGAAACTGCTGGAA 400
Query 301 GTAGGTGGCAACCCAGACAACACCCAATTCCTGTTCCTGGGTGACTATGTTGATCGTGGT 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 401 GTAGGTGGCAACCCAGACAACACCCAATTCCTGTTCCTGGGTGACTATGTTGATCGTGGT 460
Query 361 TCTTTCTCTATTGAAGTTCTCCTCCTGCTCTATGCCCTGAAAATCAACTTCCCACACAAA 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 461 TCTTTCTCTATTGAAGTTCTCCTCCTGCTCTATGCCCTGAAAATCAACTTCCCACACAAA 520
Query 421 ATCTGGCTGATCCGTGGCAATCATGAGTGCCGTCAGATGACCTCTTTCTTTAACTTTCGC 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 521 ATCTGGCTGATCCGTGGCAATCATGAGTGCCGTCAGATGACCTCTTTCTTTAACTTTCGC 580
Query 481 GACGAGTGCGAATACAAATACGACATGGTAGTCTATTACGCGTTCATGGAATCTTTCGAC 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 581 GACGAGTGCGAATACAAATACGACATGGTAGTCTATTACGCGTTCATGGAATCTTTCGAC 640
Query 541 ACCATCCCGCTCTCTGCGGTGATCAATGGTAAATTCCTCGCGGTACACGGTGGCCTCAGC 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 641 ACCATCCCGCTCTCTGCGGTGATCAATGGTAAATTCCTCGCGGTACACGGTGGCCTCAGC 700
Query 601 CCGCAACTGGTTCTGCTGAACCAAATCTGCTCTTTCACCCGTTTCCAGGAGCCACCTCGT 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 701 CCGCAACTGGTTCTGCTGAACCAAATCTGCTCTTTCACCCGTTTCCAGGAGCCACCTCGT 760
Query 661 AGCGGCATCTTCTGCGATATCCTGTGGGCTGACCCTATCGACGAAGATAAAGAGGAACAC 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 761 AGCGGCATCTTCTGCGATATCCTGTGGGCTGACCCTATCGACGAAGATAAAGAGGAACAC 820
Query 721 ACCATTCAGACCGAATCTTACTTCCCGAACGACATCCGCGGTTGCTCTTATTTCTTCGGT 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 821 ACCATTCAGACCGAATCTTACTTCCCGAACGACATCCGCGGTTGCTCTTATTTCTTCGGT 880
Query 781 TACAACGCGGCTACGACGTTCCTCGaaaaaaaCGGTCTCCTGTCTATCATCCGTGCTCAT 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 881 TACAACGCGGCTACGACGTTCCTCGAAAAAAACGGTCTCCTGTCTATCATCCGTGCTCAT 940
Query 841 GAAGCGCAACTCGAAGGCTATAAGATGCACCAAACCAACCTCAAGACCGGTTTCCCAATC 900
||||||||||||||| ||||||||| |||||||||||||||||||||||||||||| |||
Sbjct 941 GAAGCGCAACTCGAANGCTATAAGANGCACCAAACCAACCTCAAGACCGGTTTCCCNATC 1000
Query 901 GTTATCACCATTTTCTCTGCCCCGAACTACTGTGACGTTTACAACAATAAAGGTGCGGTT 960
|| |||||||||||||||||||||||||||||||||||||| || ||| |
Sbjct 1001 GTNATCACCATTTTCTCTGCCCCGAACTACTGTGACGTTTANNACNNTAANNNNNNNGNN 1060
Query 961 CTGAAATTTGACTCCAACACGCT 983
||||||||||||||| | ||||
Sbjct 1061 CTGAAATTTGACTCCNNCNCGCT 1083
Colony #2 Nucleotide Blast with DNA sequencing results (pLIC-Reverse primer)
Sequence ID: lcl|210545Length: 1232Number of Matches: 1
Related Information
Range 1: 102 to 1022Graphics Next Match Previous Match
|||||| |||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1022 TCGGTTNNNACGCGGCTACGACGTTCCTCGAAAAAAACGGTCTCCTGTCTATCATCCGTG 963
Query 836 CTCATGAAGCGCAACTCGAAGGCTATAAGATGCACCAAACCAACCTCAAGACCGGTTTCC 895
|||||||||||||||| |||||||||||||||||||||||||||||||||||||||||||
Sbjct 962 CTCATGAAGCGCAACTNGAAGGCTATAAGATGCACCAAACCAACCTCAAGACCGGTTTCC 903
Query 896 CAATCGTTATCACCATTTTCTCTGCCCCGAACTACTGTGACGTTTACAACAATAAAGGTG 955
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 902 CAATCGTTATCACCATTTTCTCTGCCCCGAACTACTGTGACGTTTACAACAATAAAGGTG 843
Query 956 CGG-TTCTGAAATTTGACTCCAACACGCTGAACATCCAACAATTCTCTTTTTCCCCGCAC 1014
||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 842 CGGNTTCTGAAATTTGACTCCAACACGCTGAACATCCAACAATTCTCTTTTTCCCCGCAC 783
Query 1015 CCGTACCACCTCCCTAACTTTATGAACCTCTTCACCTGGTCTCTCCCGTTTGTTAGCGAA 1074
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 782 CCGTACCACCTCCCTAACTTTATGAACCTCTTCACCTGGTCTCTCCCGTTTGTTAGCGAA 723
Query 1075 AAGGTTACTGAAATGCTGTATTGCATCCTGAACTCTTCTGTTAACCAGTCTGACGAAGGT 1134
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 722 AAGGTTACTGAAATGCTGTATTGCATCCTGAACTCTTCTGTTAACCAGTCTGACGAAGGT 663
Query 1135 GTAAAAGACGTAGTGCTGCCAGCGGAGGTCCTCCAGATCATCTCTTACATTGAAGAAAAC 1194
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 662 GTAAAAGACGTAGTGCTGCCAGCGGAGGTCCTCCAGATCATCTCTTACATTGAAGAAAAC 603
Query 1195 AACGTAAAACTGGAAGGTATGACCCTGAGCGGCGGTGGTGGCGCGACCGCTGGTGCAGCG 1254
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 602 AACGTAAAACTGGAAGGTATGACCCTGAGCGGCGGTGGTGGCGCGACCGCTGGTGCAGCG 543
Query 1255 TCTACCGGTGCGACCGAGGGCTCTCCGTCTTCTCAGCGTAAAGAAGCGCTGTTCAAAGAG 1314
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 542 TCTACCGGTGCGACCGAGGGCTCTCCGTCTTCTCAGCGTAAAGAAGCGCTGTTCAAAGAG 483
Query 1315 GGTTGTTTCCACTCTGGTGCGAGCAAGGAAGGTGGTGCGCTGGGCACCACTTCTCCTGCC 1374
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 482 GGTTGTTTCCACTCTGGTGCGAGCAAGGAAGGTGGTGCGCTGGGCACCACTTCTCCTGCC 423
Query 1375 GCAGCCACGGCGACTACCCAGCAGATGGCAGCTCAAGGCGAACAACCGGCGCACCTGCAC 1434
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 422 GCAGCCACGGCGACTACCCAGCAGATGGCAGCTCAAGGCGAACAACCGGCGCACCTGCAC 363
Query 1435 ACGGACGACGCGCAGGCGTCTAAAGAACGTAGCGACGCTCTGCGCAAAAAAGTACAGTCT 1494
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 362 ACGGACGACGCGCAGGCGTCTAAAGAACGTAGCGACGCTCTGCGCAAAAAAGTACAGTCT 303
Query 1495 GTCGGCCGTCTGATGCGTGTTTTCCGTACCCTCCGTAAGGAAAACGAACTGATCGTTCAA 1554
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 302 GTCGGCCGTCTGATGCGTGTTTTCCGTACCCTCCGTAAGGAAAACGAACTGATCGTTCAA 243
Query 1555 CTCAAAGGTTGCAGCCCAGGTTATCGTATCCCGGTTGGTCTGCTGCTGCAGGGTAAGGAG 1614
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 242 CTCAAAGGTTGCAGCCCAGGTTATCGTATCCCGGTTGGTCTGCTGCTGCAGGGTAAGGAG 183
Query 1615 GGTCTGGAAAATGAGCTGGAGAAATTCACCAAAGCGAAGCAAATCGACTCTATCAATGAG 1674
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 182 GGTCTGGAAAATGAGCTGGAGAAATTCACCAAAGCGAAGCAAATCGACTCTATCAATGAG 123
Query 1675 AAACGTCCGCCGAATGAGTAA 1695
|||||||||||||||||||||
Sbjct 122 AAACGTCCGCCGAATGAGTAA 102
The remaining mini-prepped sample of STPP in pNIC-Bsa4 from colony 2 was used to transform E. coli BL21(DE3) bacteria in order to prepare for protein expression.
Figure 2: Control plate. LB Agar+Kan+Sucrose plate with untransformed E. coli BL21(DE3) bacteria.
There are no colonies on the control plate. This indicates that sterile technique was good because the Kanamycin (an antibiotic) killed the untransformed cells because they lack Kanamycin resistance.
Figure 3: BL21(DE3) transformed with P. vivax STPP grown on LB+Kan+Suc plates. 10 uL of the final transformation mixture was plated.
There are very few colonies on this plate, which indicates that 10 uL may have been too small an amount to plate for optimal growth of the bacteria.
Figure 4: BL21(DE3) transformed with P. vivax STPP grown on LB+Kan+Suc plates. 50 uL of the final transformation mixture was plated.
There are many more colonies on this plate than were on the 10 uL plate. This indicates that adding 50 uL of the transformation mixture was much more desirable for bacterial growth.
More DH5-Alpha with STPP in pNIC-Bsa4 from colony #2 on the master plate were grown up in an overnight culture, spun down, and midi-prepped.
Figure 5: Concentration of midi-prepped P. vivax STPP in pNIC-Bsa4. Conc= 68.4 ng/uL.
This midi-prepped plasmid can be used in the future to transform more BL21(DE3) as needed. Midi-prepping is cleaner than mini-prepping, so this sample is likely cleaner than the mini-prepped sample used for the first round of transformation of BL21(DE3).
Figure 6: Concentration of purified P. vivax STPP Elution 1. Conc= 0.46 mg/mL.
FIgure 7: Concentration of purified P. vivax STPP Elution 2. Conc= 0.23 mg/mL.
Summary/Analysis:
Elution 1 of the purified protein is more highly concentrated than Elution 2, which is to be expected. Elution 2 is the remained of the protein that is eluted for a second time from the Ni-NTA resin. Samples were taken throughout the purification process and SDS-Page gel will be run to characterize the protein and determine its purity. If protein is impure, FPLC will be done.
Week 5 &6
- Nice work. Try to include some analysis of your data. Thank you. -Max 10/07/2013
Another attempt was made to grow up DH5-alpha with pNIC-Bsa4. Again, the yield was very small. Transformation of DH5-alpha cells with the pNIC-Bsa4 plasmid was done again in order to get new colonies from which to grow the bacteria.Figure 1: Nanodrop reading for midi-prepped pNIC-Bsa4 plasmid. Concentration is 55.3 ng/uL.
Figure 2: Nanodrop reading (Trial 1) of cut plasmid pNIC-Bsa4. Concentration is 8.2 ng/uL.
The concentration of the cut plasmid is very low.
Figure 3: Nanodrop reading (Trial 2) of cut and PCR cleaned plasmid pNIC-Bsa4. Concentration is 151.8 ng/uL.
Three tubes were prepared, each with total volume of 50 uL. All samples were mixed into one tube during PCR cleanup, and the plasmid was eluted into 30 uL of buffer.
Figure 4: Plasmodium vivax serine-threonine phosphatase, catalytic subunit 2B gene in plasmid vector pNIC-Bsa4 in DH5-Alpha cells. Plates are LB+Kan+Sucrose. Cloned with 2 uL accepting vector (pNIC-Bsa4) and 4 uL gene insert.
Figure 5: Plasmodium vivax serine-threonine phosphatase, catalytic subunit 2B gene in plasmid vector pNIC-Bsa4 in DH5-Apha cells. Plates are LB+Kan+Sucrose. Cloned with 2 uL accepting vector (pNIC-Bsa4) and 8 uL gene insert.
Figure 6: Master plate for Plasmodium vivax serine-threonine phosphatase, cataytic subunit 2B gene in pNIC-Bsa4 plasmid vector in DH5-Alpha cells. Plates are LB+Kan+Sucrose.
Summary/analysis:
The first cloning attempt was made for this target. Bacterial colonies were present on the LB+Kan+Sucrose plates for both the original plate and the master plate, which indicates that the SacB gene is not in these bacteria. Mini-prep, RE digest, and sequencing will be performed next week in order to determine if these are positive clones.
Week 3 & 4
Figure 1: Secondary PCR for Plasmodium vivax serine-threonine phosphatase, catalytic subunit 2B, putative gene.
Lane 1: 1 kb ladder
Lane 5: Secondary PCR (using primary PCR as template)
Lanes 2-4 were PCR samples of another researcher.
Analysis: Lane 5 does not show a distinct band, so the secondary PCR was unsuccessful.
Figure 2: Secondary PCR (Trial 2) of Plasmodium vivax serine-threonine phosphatase, catalytic subunit 2B, putative gene.
Lane 1: 1 kb ladder
Lane 2: empty
Lane 3: Secondary PCR (using primary PCR as template)
Analysis: Lane 3 shows a distinct band between 1.5 and 2.0 kilobases. The target gene is 1,977 bp, so the PCR seems to be successful. For this PCR, the annealing temperature used was 59 degrees C, and the time for annealing and elongation was increased to 30 seconds each.
Figure 3: PCR2 of Plasmodium vivax serine-threonine phosphatase, catalytic subunit 2B, putative gene.
Lane 1: 1 kb ladder
Lane 2: Secondary PCR (Ariel C.)
Lane 3-6: PCR2 reaction with identical amounts of reagents
Analysis: Lanes 3-6 show very distinct bands between 1.5 and 2.0 kb. Because the P. vivax STPP gene is 1,977 bp, it can be concluded that the PCR was successful in amplifying the gene. Annealing temperature: 59 degrees.
Figure 4: Nanodrop reading after PCR cleanup on PCR2 product for Plasmodium vivax serine-threonine phosphatase, catalytic subunit 2B, putative gene.
Analysis: The 260/280 and 260/230 values are very close to 2, which indicates that the DNA sample is pure.
Figure 5: Nanodrop reading of pNIC-Bsa4 plasmid (from DH5-alpha) after midi-prep.
Analysis: The nano drop reading shows a negative yield for the plasmid, which indicates that the plasmid was not in the midi-prepped sample. The cell pellet from the overnight culture of DH5-alpha was very small (even after several trials), which indicates that there is a problem with the plate. Another overnight culture will be prepared for midi-prep.
Summary:
Secondary PCR and PCR^2 reactions were completed for P. vivax serine-threonine phosphate, catalytic subunit 2B, putative. A Nanodrop reading was taken and the sample was found to be relatively pure. Cells of DH5-alpha transformed with the pNIC-Bsa4 plasmid were midi-prepped, but there was no plasmid yield. The next step is to grow more overnight culture of DH5-alpha and perform midi-prep again.
Week 1 & 2
Figure 1: Primary PCR for Plasmodium vivax serine-threonine phosphatase, catalytic subunit 2B, putative.
Lane 1: 1 kb ladder
Lane 9: Primary PCR (PCR on oligo mix for P. vivax STPP 2B)
Lanes 2-8 were PCR samples of other researchers.
Summary:
This week I made an oligo mix for the primers that will form the gene which codes for P. vivax serine-threonine phosphatase, catalytic subunit 2B, putative. I then did a primary PCR with the oligo mix.
I grew an overnight culture of pNIC-Bsa4 in DH5-Alpha. The cells have been spun down in a centrifuge. These will be midi-prepped in the future.
Analysis:
The "smear" that appears on Lane 9 of the gel indicates that the primary PCR was successful. The smear should be composed of several separate strands of DNA that will be put together with secondary PCR.
Fall 2013
WEEK 4
Figure 1: RE digest of pGBR22 using Restriction enzymes EcoRI-HF and PvuII (left side of gel)
Lane 1: empty
Lane 2: 1 kb ladder
Lane 3: Uncut plasmid
Lane 4: pGBR22 with EcoRI-HF in NE buffer 2
Lane 5: pGBR22 with PvuII in NE buffer 2
Lane 6: pGBR22 with PvuII and EcoRI-HF in NE buffer 2
Lane 7: pGBR22 with EcoRI-HF in NE buffer for EcoRI
Absorbance measurement of yopH Elution 1 at 280 nm. Absorbance is 0.940.
Absorbance measurement of yopH Elution 2 at 280 nm. Absorbance is 0.109.
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WEEK 3
Figure 1: Trial 1 PCR of pGFP. DNA failed to amplify
Lane 1: 1 kb ladder
Lane 2: Sample A, VDS primers
Lane 3: Sample B, VDS primers
Lane 4: Sample C, VDS primers
Lane 5: 1 kb ladder
Lane 6: Sample D, VDS primers
Lane 7: Sample E, M13 primers
Lane 8: Sample F: M13 primers
Lane 9: Sample G, M13 primers
Lane 10: Sample H: M13 primers
Figure 1: Trial 2 PCR of pGFP. DNA may have amplified in lane 4, but PCR needs to be redone with 100 bp ladder.
Lane 1: 1 kb ladder
Lane 2: Sample A, VDS primers
Lane 3: Sample B, VDS primers
Lane 4: Sample C, VDS primers
Lane 5: Sample D, VDS primers
Lane 6: 1 kb ladder
Lane 7: Sample E, M13 primers
Lane 8: Sample F: M13 primers
Lane 9: Sample G, M13 primers
Lane 10: Sample H: M13 primers
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WEEK 2
Figure 1: PCR of plasmid pGBR22 (left side of gel).
Anita Vasudevan 6/13/2013 VDS
PCR of plasmid: pGBR22
Lane 1: 1 kb Ladder
Lane 2: Sample A, 1:500 template DNA, 1 uL Taq
Lane 3: Sample B: 1:500 template DNA, 1 uL Taq
Lane 4: Sample C: 1:50 template DNA, 1 uL Taq
Lane 5: Sample D: 1:50 template DNA, 1 uL Taq
Figure 2: Nanodrop spectrometer Run 1 reading for puc19 plasmid purified from E.coli (DH5-alpha). Concentration is 49.0 ng/uL
Figure 3: Nanodrop spectrometer Run 2 reading for puc19 plasmid purified from E. coli (DH5-alpha). Concentration is 41.4 ng/uL
WEEK 1
Figure 1: E. coli (DH5-alpha) cells transformed with 1 ng puc19 plasmid grown on LB+Amp Agar plates.
To count colonies:
~divided into 45 equal squares
~approx. 28 colonies per square
~(45*28)= 1,260 colonies
Transformation efficiency: 1,260 colonies/1 ng= 1,260 colonies/ng
Figure 2: E. coli (DH5-alpha) cells transformed with 5 ng puc19 plasmid grown on LB+Amp Agar plates.
To count colonies:
~divided into 45 equal squares
~approx. 67 colonies per square
~(45*67)= 3,015 colonies
Transformation efficiency: 3,015 colonies/5 ng= 603 colonies/ng
Figure 3: E. coli cells (DH5-alpha) transformed with 25 ng puc19 plasmid grown on LB+Amp Agar plates.
To count colonies:
~divided into 45 equal squares
~approx. 12 colonies per square
~(45*12)= 540 colonies
Transformation efficiency: 540 colonies/25 ng= 21.6 colonies/ng
DNA sequencing (M13 Forward)
Nanodrop spectrometer ("purple" plasmid: gbr22)